doi: 10.1242/10.1242/dev.00503
Targeted ablation of CCAP neuropeptide-containing neurons of Drosophila causes specific defects in execution and circadian timing of ecdysis behavior
Jae H. Park1,*,
Andrew J. Schroeder2,*,
Charlotte Helfrich-Förster3,
F. Rob Jackson2 and
John Ewer4,
1 Department of Biochemistry and Cellular and Molecular Biology, University of
Tennessee, Knoxville, TN, and Department of Biology, Brandeis University,
Waltham, MA, USA
2 Department of Neuroscience, Tufts University School of Medicine, Boston, MA,
USA
3 Zoological Institüt, University of Tübingen and Regensburg,
Germany
4 Cornell University, Entomology Department, Ithaca, NY, USA
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Fig. 7. Anatomy of CCAP neurons and their role in the clock control of eclosion.
(A,B) overlap among the projections and arborizations of CCAP-,
timeless-, and PDF-containing neurons. (A) Overlap between
projections (arrow) of protocerebral CCAP neurons and tim-containing
DN2 neurons of the 3rd instar larval brain. (B) Overlap of the synaptic fields
(arrow) of tritocerebral PDF (green) and subesophageal CCAP neurons of the
pharate adult brain. Asterisk shows ending of the LN[v] clock cells. DN2,
dorsal neuron 2; Tri, tritocerebrum. POT, posterior optic tract (containing
projections of the LN[v] clock cells). (C,D) Eclosion profiles for KO (black
bars) and control (gray bars) populations under (C) LD (12 hours light:12
hours dark) and (D) DD (continuous darkness). The profile for subjective days
4 and 5 is shown. Height of each bar represents the percentage of flies that
eclosed within a 2 hour window normalized to the total number of flies that
eclosed during that day (indicated separately for each genotype and day). The
open and closed horizontal black rectangles (C) show the light and dark phases
of the LD schedule, respectively, while the black and gray rectangles in D
show those for the LD regime prior to the shift to DD. Collections were
performed at the zeitgeiber (ZT) or circadian (CT) times indicated.
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Fig. 1. Nucleotide and deduced amino-acid sequence of the DmCCAP gene. (A)
Genomic sequence of DmCCAP. Sequences for the partial
5'-upstream region and for the cDNA are indicated in upper case, and the
intervening sequences are presented in lower case. A consensus polyadenylation
signal (AATAAA) is underlined, and the transcription initiation site is
designated by a bold-faced letter. A putative arthropod initiator (TCATT) and
a downstream promoter element (GTCG) are shaded gray. A putative signal
peptide is indicated by italics; amino acids represent the predicted
pre-pro-DmCCAP peptide. Potential endoproteolytic cleavage sites are
designated by asterisks. (B) Schematic diagram of the genomic organization of
DmCCAP. Open boxes represent exons and solid lines represent introns.
Numbers indicate the nucleotide length for the corresponding exons and
introns. Approximate positions for the start (ATG) and stop (TAA) codons are
indicated by arrows. (C) Reconstruction of the pre-pro-DmCCAP structure. SP,
signal peptide; CCAP-AP I, II, and III: CCAP-associated peptides I, II and
III, respectively. CCAP and the other domains are represented by a shaded box
and by open boxes, respectively. The number in each box indicates the amino
acid length for each domain. The consensus endoproteolytic cleavage sites are
also shown between the boxes. (D) Comparison of the amino acid sequences of
CCAP precursors. Manduca sexta sequence from Loi et al.
(Loi et al., 2001 ); mosquito
(Anopheles gambiae) CCAP gene sequence was obtained from mosquito
genome project database (agCG50022: accession no. EAA14174). Identical amino
acids are highlighted in bold; there is a perfect match between sequences for
the CCAP peptide (underlined). In addition, a significant homology was
observed for the CCAP-AP III predicted peptide. Consensus proteolytic cleavage
site between DmCCAP-AP II and III was not found in Manduca CCAP
precursor structure. Surprisingly, the amidation signal (GRKR) was absent from
the mosquito sequence, suggesting that CCAP in this insect may not be modified
at its C terminus, resulting in much longer CCAP-like peptide. More careful
characterization of the corresponding cDNA will be necessary to confirm this
result.
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Fig. 2. Expression of CCAP RNA in CCAP neurons, and use of CCAP-GAL4
fusion for targeted ablation of CCAP neurons. (A,B) Expression of CCAP-IR (A)
and of CCAP RNA (B), in 3rd instar larva CNS. Neurons located in similar
positions are indicated by the same symbols, emphasizing the similarity of the
two patterns of expression. (C,D) CCAP-IR (brown) in combination with CCAP RNA
expression (blue), illustrating co-localization of these two signals in the 2
pairs of CCAP-immunoreactive neurons in the brain (arrowheads in A and B). (D)
Higher magnification of boxed pair of neurons in C; the 2 neurons are very
close to each other. Arrowheads point to (clear) nuclei; blue staining is due
to RNA expression in the cell bodies, while brown is CCAP-IR, and is
especially visible in the neuronal processes (asterisk in C and D). (E)
Pattern of CCAP-IR (red and upper right panel) and ß-gal-IR (green and
lower right panel) in late 2nd instar CNS of CCAP-GAL4 x
UAS-lacZ progeny. All CCAP-immunoreactive neurons were ß-gal
immunoreactive, and vice versa. (F) Targeted ablation of CCAP neurons. Pattern
of CCAP-IR (red) and ß-gal-IR (green) in CNS of CCAP-GAL4;
UAS-rpr + lacZ late 2nd instar. Br, brain; vns, ventral
nervous system. Scale bar: (A) 80 µm, (E) 40 µm.
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Fig. 3. Larval ecdysis behavior in KO animals. (A) Timing of morphological and
behavioral markers at ecdysis from the 2nd to the 3rd larval instar. (B)
Direct comparison of the duration of pre-ecdysis (a) and of the components of
ecdysis (b). Open bars show the period between DMP and onset of pre-ecdysis
(gray bar). `Air' marks the time of entry of air into the 3rd instar trachea.
Pre-ecdysis terminates with the occurrence of `biting' behavior (B), which is
then followed by the expression of posterior-to-anterior peristalses (P/A) and
the ensuing shedding of the old cuticle (E, ecdysis). Records were aligned
relative to the time of tracheal air filling (`Air') and are
averages±s.e.m.; n=8 for each group. These data and the
statistical significance of the observed differences are given in
Table 1.
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Fig. 4. Targeted ablation of CCAP neurons causes failures of pupation. (A-D) Early
KO pupa (A,B) and corresponding control (C,D). KO animals show defects such as
incomplete head eversion, as evidenced by the anterior position of head (white
asterisk indicates the position of the eye); retracted posterior cuticle due
to failure to transition from pre-ecdysis to ecdysis (arrow in A), and
incomplete shedding of tracheal lining (arrowheads in B point to scars left on
the larval cuticle). White arrows in D indicate properly everted legs (l) in
control animal. (E-H) KO (E-G) and control (H) pharate adults. The failure at
pupation in KO animals results in defects in adult head formation and in leg
and wing extension. Black asterisk: partial adult head; m, larval mouthooks;
p, proboscis of adult. White arrows and black arrowheads in E-H indicate the
posterior extent of the wings and prothoracic set of legs, respectively. (I)
Timecourse of morphological and behavioral events at pupation. Pharate pupae
collected at late stage P4(i) (Bainbridge
and Bownes, 1981) were first quiescent (green gradient), then went
into pre-ecdysis (yellow), followed by ecdysis (head eversion; red segment),
which was specifically absent in KO animals. Ecdysis (in the control) and the
extended pre-ecdysis (of KO animals) were followed by a long period of
abdominal movements (blue gradient). Records were aligned relative to the
onset of pre-ecdysis, and the duration of each interval is indicated as
average±s.e.m.; times prior to pre-ecdysis were not tabulated. These
data and the statistical significance of the observed differences are
tabulated in Table 2.
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Fig. 5. CCAP and ETH are released at pupation. (A) Pattern of CCAP-IR in the CNS of
a pre-pupa. (B-G) Enlargement of boxed area in A, showing (B-D) CCAP-IR in
descending axons (arrows) and (E-G) ETH-IR in Inka cell before pre-ecdysis
(B,E), at start of pre-ecdysis (C,F) and immediately after (D,G) pupal
ecdysis. (H) Quantitation of the intensity of CCAP-IR in descending axon
(arrow in B-D) and of ETH-IR. Before, before pre-ecdysis (as in B,E); Pre, at
the start of pre-ecdysis (as in C,F); Ecd, immediately after pupal ecdysis (as
in D,G). Values are averages±s.e.m.; 8-10 preparations were scored for
each time point. Scale bars: 40 µm (D); 10 µm (G).
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Fig. 6. Targeted ablation of CCAP neurons causes defects at eclosion and in
post-eclosion events. (A,B) Pharate KO (A) and control (B) animals at
eclosion. While both flies inflated their ptilinum (arrow), the abdomen of KO
pharate adults failed to distend and exert traction on the internal surface of
the puparium. White arrowheads indicate outer limits of abdomen; black bars
indicate width of puparium. (C) Adult KO animals did not inflate wings (arrow)
or correctly tan the cuticle. White arrows indicate dimples in dorsal thorax
at insertion point of thoracic musculature. (D) Control adult of similar age
(2- to 4-day old). (E) Eclosion in control and KO animals. Eclosion behaviors
(red bars; triangles represent 10 bouts of eclosion peristalses) were preceded
by the entry of air into the trachea (Air) and by the deployment of the
ptilinum (EP). Successful extrication from the puparium is indicated
(Eclosion). Times represent the average length of each interval±s.e.m.;
n=10 for each group, except for eclosion events themselves where
n=9 for KO animals (one animal of the 10 animals monitored failed to
escape the puparium). The most noticeable difference between KO and control
pharate adults was that the former showed many more bouts of eclosion behavior
before emerging from the puparium, due primarily the poor traction exerted by
the abdomen on the inside walls of the puparium as seen in A.
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© The Company of Biologists Ltd 2003
